Both chemical and genetic inactivation of Cys38-S− completely abrogated ALV fusion and infection. Remarkably, Cys38-S− does not mediate isomerization of the SU-TM disulfide bond and is not required for Tva-induced activation of TM, including pre-hairpin association with membranes and low pH assembly of helical bundles. These findings indicate that, contrary to current models, receptor activation of TM is not sufficient for ALV fusion and infection and that formation of a reactive thiolate is an additional receptor-dependent step.According to current models of retrovirus infection, receptor binding to the surface subunit (SU) of the envelope glycoprotein (Env) triggers a conformational change in the transmembrane subunit (TM) that mediates virus fusion to cell membranes. To understand how this occurs, we investigated the role of the receptor Tva in avian leukosis virus-A (ALV-A) infection. We find that Tva binding induced the formation of a reactive thiolate on Cys38  of these viruses are activated by their encounter with specific receptor proteins on target cells. In this scheme, receptor binding to the glycoprotein surface subunit releases constraints on the intrinsic membrane fusion activity of the transmembrane subunit. New studies summarized in this report have examined the chemical basis for receptor activation of avian retrovirus envelope glycoproteins. They show that receptor binding creates a reactive cysteine thiolate in the surface subunit that is essential for infection. Remarkably, this cysteine thiolate is not required to activate the known steps in infection mediated by the transmembrane subunit, including insertion into the target membrane and assembly into helical bundles. Thus, the receptor-induced cysteine thiolate mediates a key step in infection that is not accounted for by current models of Env function. Enveloped virus infection is mediated by glycoproteins that protrude from the virus membrane. There are striking structural and functional similarities among the envelope glycoproteins (Envs) of ortho-, paramyxo-, retro-, filo-, and coronaviruses. As such, they are considered together as class I Envs . Class IIn addition to stabilizing pre-fusion TM, the membrane distal subunit is also the sensor for the physiological factors that trigger infection. For retroviruses, these factors are membrane proteins or receptors that bind to the membrane distal or surface subunit (SU). Discrete receptors have been identified for nearly all classes of retroviruses, and the basis for receptor-Env binding has been extensively analyzed . These s−) in SU that is required for infection. Remarkably, in the absence of the critical Cys-S−, membrane fusion and infection are uncoupled from TM activation, thus revealing a previously unidentified role for SU in ALV infection.For avian alpharetroviruses, a structural correlate is lacking; however, there is functional evidence for a multi-step triggering mechanism . In partThe thiol-specific alkylating reagent, PEO-maleimide biotin (PMB), was used to probe for functionally important thiolates in ALV-A. Purified ALV-A virus particles were incubated with PMB for 30 min at 37 °C, and infectivity was measured on human 293 cells expressing the ALV receptor Tva (293-Tva). PMB treatment alone had little or no effect on ALV-A infection under these conditions. However, exposure of ALV-A particles to PMB (10 mM) in the presence of sTva, a functional isoform of the ALV-A receptor lacking the transmembrane domain, reduced infection by >99% . In cont−) target for PMB in ALV-A Env. The marked sensitivity of the chimeric particles to PMB inhibitor compared to ALV-A may reflect poor incorporation of ALV-A Env into MLV particles and/or a synergistic effect of PMB on ALV-A Env and MLV core.Because PMB is membrane impermeable and its inhibitory activity is sTva-dependent, the suspected PMB target is the ALV-A Env glycoprotein. To test this hypothesis, we studied chimeric virus particles in which ALV Env was expressed on MLV particles. The effect of PMB treatment of these particles was comparable to native MLV particles. However, PMB treatment in the presence of sTva reduced infectivity to less than 0.001% . These f− by PMB is irreversible. To identify the PMB target, Env was recovered from lysates of PMB-treated ALV-A by immunoprecipitation using an anti-TM antibody and reduced and denatured subunits were probed for the biotin moiety using streptavidin-HRP. Viruses exposed to 1 mM sulfo-NHS-biotin (SNB), which reacts with primary amines, served as a control. SU and TM were the predominant targets for covalent modification by SNB , in the presence of sTva. NEM-modified SU was purified from virions, and N-linked carbohydrates were removed using PNGase F. The deglycosylation reaction products were resolved by SDS-PAGE and quantified by SYPRO Ruby staining B. The poenv genes bearing individual Cys to Ser substitutions were inserted into an ALV-A provirus, RCASBP(A), that encodes enhanced green fluorescent protein (EGFP) as a marker for infection. Each provirus was then transfected into avian DF-1 cells, and the production of replication-competent virus was monitored by the spread of EGFP expression in the culture. Viruses bearing the C60S or C91S substitutions were infectious and indistinguishable from wild type ALV in all assays and were not studied further  in ALV-A infection occur after receptor binding.The role of Env Cys 38 in ALV-A infection was pursued. To test whether this residue is necessary for SU binding to receptor, immunoadhesins comprised of wild type or Cys38Ser ALV-A SU fused in-frame to the constant region of a rabbit immunoglobulin gamma chain (SUA-rIgG) were produced in 293 cells, purified by protein A affinity chromatography, and used as ligands. The binding profile of Cys38Ser SUA-rIgG for 293-Tva cells is indistinguishable from wild type SUA-rIgG protein . NeitherAccording to current models, exposure of the Tva-bound Env to endosomal acid pH alters inter-subunit contacts and releases metastable TM to undergo the conformational change that is coupled to membrane fusion. In particular, the driving force for fusion is supplied by formation of a highly stable helical bundle. The bundle is formed by a hairpin turn in each TM such that the membrane proximal segments fold back into the grooves on the outer surface of an extended three-stranded coiled coil. Previous studies have shown that helical bundle formation can be monitored by the formation of an SDS-resistant TM oligomer ,15,16. WTo confirm that the SDS-resistant oligomer is indicative of a post-fusion conformation of TM, we tested the ability of the R99 peptide inhibitor of ALV infection to block its formation. The R99 peptide is derived from the HR2 segment of TM and inhibits formation of the helical bundle by competing with HR2 for binding to grooves on the outer surface of the central three-stranded coiled coil ,18. Aliq− is a determinant of Env stability, the effect of Cys38Ser on the temperature threshold for oligomer formation was measured. Virus particles were incubated alone or in the presence of sTva (100 nM) at temperatures from 45 °C to 65 °C. After 30 min, the particles were rapidly cooled to room temperature and the conformation of TM was analyzed. The findings demonstrate that the 60 °C threshold for heat-induced oligomer formation is not altered in mutant Env . These findings indicate that Cys38-S− does not catalyze isomerization of the SU-TM disulfide bond.Like ALV, a critical reactive thiol has also been identified in MLV SU. Several studies have provided evidence that the essential function of the critical MLV thiol is as a nucleophile in receptor-induced isomerization of the disulfide bond linking SU and TM ,21. A po− is the active site of an intrinsic cysteine protease that resides in ALV Env, we measured ALV-A infection in the presence of E-64. E-64 is a broadly active cysteine protease inhibitor that inactivates host cysteine proteases and blocks infection by the Env GP of Ebola virus [− is not the active site of papain-like protease activity that is required for infection.Reactive cysteine thiolates in extracellular proteins also function as the active sites of proteases, including endosomal cysteine proteases. Cleavage of Env by endosomal cysteine proteases is required for Ebola and SARS infection ,23. The la virus . E-64 tr− activity is necessary for virus association with target membranes, perhaps by alleviating a steric block imposed by SU. To address this question, the requirements for wild type and mutant ALV-A particles to associate with liposome targets was measured. Purified ALV-A particles were incubated with liposomes in the presence of sTva for 20 min on ice  and then for 30 min at 37 °C at pH 8.0. The incubation mixture was divided, and each aliquot was treated to adjust the pH from 7.4 to 5.0. After 30 min at 37 °C, the pH of the sample was brought to 7.5 and formation of virus-liposome complexes was assessed by density gradient centrifugation. During centrifugation, the liposomes migrated to the top of the gradient where they are visible and virions (located by immunoblot for virus capsid p27) remained at the bottom of the gradient . We also determined the conformation of TM in these samples. Remarkably, we observed that that PMB abrogated the low pH requirement for TM bundle formation A. The ch− B. Althou− target that is inaccessible or unreactive in native Env. This conclusion is consistent with previous reports that Tva binding enhances the sensitivity of ALV SU to thermolysin cleavage [The entry of retroviruses into cells is initiated by binding of virus Env to cell surface receptor proteins. Each class of retroviruses uses one or in some cases a few closely related cell surface proteins as receptors. Although receptors have been identified for most retroviruses, how binding to these proteins triggers infection is poorly understood. ALV is an excellent model to study this problem because well-characterized assays of receptor function have been established. The hypothesis tested in this report was suggested by an analysis of cysteine residues in Env. In the mature glycoprotein, a single disulfide bond joins SU and TM, and SU-SU disulfide bonds are not present; therefore, at least one of fourteen cysteine residues in SU lacks a partner to form an intramolecular disulfide bond. We used thiol-reactive alkylating agents to probe for the unpaired thiols in SU and found that this reaction is Tva-dependent. This indicates that receptor binding induces a conformational change in SU that creates a CysScleavage ,29.−. Determination of the redox state of the Cys thiols in ALV Env before and after binding to Tva is needed to ascertain the relationship between receptor binding and Cys38-S− formation. The most straightforward possibility is that Cys38-S− is present in mature Env, but is inaccessible or unreactive to thiol alkylating agents. Alternatively, Cys38-S− may be the product of intramolecular disulfide isomerization initiated by another Cys-S− in SU, including Cys292 and/or Cys309. Because the number and distribution of cysteine residues including Cys38 is conserved in Envs of ALV subgroups B, D, E and J, receptor-induced formation of reactive Cys-S− residues in SU may be an essential property of all alpharetroviruses.Remarkably, ALV infection was markedly inhibited by alkylation of thiols on SU. Analysis of Env identified three Cys residues with thiols that are targets and further studies of one these residues, Cys38, was highly informative. Infection by mutant ALV particles lacking Cys38 was never observed and reversion of these particles to wild type did not occur during prolonged cultivation of virus producing cells. Although Cys38 is essential for infection, it has no apparent role in maturation of pre-fusion Env. In addition, replacement of Cys38 with serine did not alter the affinity of SU for Tva or the temperature threshold for heat-induced folding of virus TM into the SDS-resistant oligomers containing the helical bundle. The temperature threshold for heat-induced bundle formation is a particularly sensitive indicator that the intersubunit interactions that stabilize the metastable pre-fusion conformation of Env are not dependent on Cys38 ,6. Based− mediates a post-receptor step. Current models indicate that the critical post-receptor step is activation of TM [−-dependent.The absence of a detectable influence of Cys38 on Env maturation and the receptor-dependence of reactive thiol strongly suggests that Cys38-Son of TM –4,8. Afton of TM ,6. From on of TM . Tva binon of TM ,25. Tva on of TM , includion of TM –18. Usin− in helical bundle formation. In this assay, brief exposure to pH 5.0 or to 5.5 was associated with apparent increase in the density of sTva-induced virus-liposome complexes, perhaps due to a reduction in the number of liposomes associated with each particle. Alternatively, this change in density may reflect the dissociation of capsid from the liposome-associated particle. This increase in liposome-associated particle density induced by acid pH is dependent on bundle formation, since the R99 inhibitor of infection specifically blocked the acid pH-dependent shift in density. Although the molecular basis for the shifting mobility of virus particles is unclear, it is another parameter that is closely correlated with bundle formation and infection, yet conserved in the absence of Cys38-S−. Taken together, these studies support the conclusion that activation of TM is not the essential function of Cys38-S− in infection.Analysis of the migration of virus/liposome complexes in sucrose density gradients provided another parameter to investigate the potential role of Cys38-S− -dependent steps in Env function that are not accounted for in current models of fusion? One possibility is that SU might sterically interfere with correct insertion of TM into the membrane unless removed. For HIV, gp120 is non-covalently linked to gp41, which may facilitate the displacement of gp120 from the site of fusion [−. So, a simple model of SU release is not supported. Moreover, a strict requirement for the release of the membrane distal subunit for Type I Env infection is excluded because the covalent interaction between HA1 and HA2 is not disrupted during influenza infection [− to promote infection by disrupting the covalent association between SU and TM.What are possible Cys38-Sf fusion . Like ALf fusion . In cont− results in changes in TM activation that are not detected by our assays. For example, the fusion-active pathway could be dependent upon synchronous Env activation mediated by Cys38-S−. Thus, a decrease in the rate of helical bundle formation in the mutant Env could shunt TM into a dead-end pathway that does not result in fusion. If true, the difference in active and dead-end pathways must be subtle, since Cys38 did not measurably alter the threshold for thermal activation of Env/Tva complexes. Future studies directed at comparing the structure and kinetics of Env activation in native and in Cys38-deficient particles are needed.A second possibility is that the absence of Cys38-S− is essential is that it mediates a key step that occurs after bundle formation. Previous studies revealed close spatial and temporal relationships between bundle assembly and membrane merger/pore formation [− activity promotes expansion of the nascent pore, possibly through direct reaction with the membrane. Although speculative, this hypothesis is testable using more sensitive measures of lipid and content mixing. However, the failure of chlorpromazine to rescue the fusion defect of the Cys38Ser mutant suggests that fusion is blocked prior to hemifusion.A straightforward explanation for why Cys-38-Sormation ,32. In pormation . In addiormation . Therefo− in infection is to identify its substrate. Based on analogy to MLV [− may participate in isomerization of intramolecular disulfide bonds within SU that is necessary for proper timing or alignment of TM activation for efficient fusion.The observation that thiol alkylation inhibits infection is consistent with an intrinsic enzymatic activity in SU. In extracellular proteins, the majority of functionally important thiolates form the active sites of enzymes, and cysteine-alkylating agents are potent, irreversible inhibitors of these enzymes. Therefore, an alternative approach to identify the role of Cys38-Sy to MLV  and pesty to MLV , a leadi− is the active site of intrinsic Env protease activity. Indeed, a role for host cysteine protease activity is identified for filovirus and coronavirus infection [− is part of the active site of phospholipase or forms thioester adducts with fatty acids in the target membrane. Phospholipase activity has been observed in membrane disruption by parvovirus [Another possibility is that Cys38-Snfection ,23. We frvovirus . And, th− to other thiols. We found two other receptor-induced thiolates at positions 292 and 309. The reactivity of these thiolates is independent of Cys38-S−, since Tva-dependent modification occurred in the absence of Cys38. An unusual property is that NEM modification of these residues induced bundle formation. Unfortunately, their role is less well defined since mutants were unable to fold and could not be studied. This suggests that, unlike Cys38, they participate in one or two structurally important disulfide bonds (either by being bonded to each other or to unidentified partners), and that their reactivity is generated by reduction of these bonds. The thiolate groups of these residues may be titrated by low pH as part of TM activation, since both low pH and NEM modification are correlated with bundle formation. These studies provide more evidence that in the absence of Cys38-S−, bundle formation alone is not sufficient for fusion and infection. Further studies are needed to clarify the states of the thiols in SU in the pre- and post-receptor states to fully understand the role of Cys38-S− activation in infection.A key to future studies may be in understanding the relationship of Cys38-S2. The growth medium of 293-Tva cells was supplemented with 1 μg/ml puromycin.Human 293 cells were obtained from the ATCC. Avian DF-1 cells that produce a replication-competent ALV-A encoding enhanced green fluorescent protein (RCASBP(A)-EGFP, DF-1-ALV-A), a 293-derived cell line that expresses Tva (293-Tva or 2.1), and a 293-derived cell line that expresses mCAT-1 (293-mCAT) have been previously described . These cN-ethyl maleimide (NEM) was obtained from Sigma. PEO-maleimide biotin (PMB) and Sulfo-NHS-biotin (SNB) were obtained from Pierce.Affinity purified, polyclonal rabbit antisera against the C-terminus of TM (TM-1) has been described previously . Goat ang (max) in a SW28 swinging bucket rotor (Beckman). The virus pellet was re-suspended in HN buffer  for 1 hour at 4 °C before use.DF-1-ALV-A cells were grown in roller bottles with extended surface area in 50 mls of medium. Supernatants were harvested twice a day, and individual collections were pooled until 1 L of supernatant accumulated. Supernatants were pre-cleared by low speed centrifugation and filtered through 0.45-μm filters to remove cells and cell debris. Aliquots were frozen for later use. Virus was purified and concentrated by pelleting through a sucrose cushion  for 1 hour at 4 °C at 113,000 x Plasmids encoding Friend MLV Env, MLV gag-pol, and MLV LTR-EGFP have been described . To geneE. coli as previously described [sTva was purified from escribed .g in an Allegra 6R centrifuge (Beckman) at 4 °C and then centrifuged for an additional 30 minutes while the temperature was gradually raised to 35 °C as previously described. Plates were then transferred to a 37 °C incubator and cultured for 36–48 hours before being fixed in paraformaldehyde and analyzed by flow cytometry for EGFP expression.Virus was concentrated from supernatants by two cycles of ultracentrifugation through a 15% sucrose cushion in HN buffer, resuspended in a small volume of HN for 1 h at 4 °C, and incubated with 10 μM sTva to allow binding. PMB was dissolved in HN buffer and diluted into the virus/sTva samples to the indicated final concentrations. The pH of the concentrated solutions (pH 8.0) was verified prior to addition to the virus. Samples were incubated at 37 °C for 30 min, cooled on ice, serially diluted in growth medium supplemented with 40 mM HEPES, and added on ice to 12-well plates of 293-mCAT (for MLV infection) or 293-Tva cells (for ALV-A and chimeric ALV/MLV infections) that were pre-chilled to 4 °C. Plates were centrifuged for 2 hours at 1640 x 2 (HNC) and incubated for 20 minutes on ice with the indicated concentration of sTva to allow binding, for 30 min at 37 °C at pH 8.0, and then for 30 min at 37 °C at pH 5.0 or 8.0 in pre-determined volumes of 100 mM Tris-acetate or Tris-HCl. Where indicated, 1 mM PMB was added to the samples after the 20 min incubation with sTva on ice. The samples were then neutralized with 1 M HEPES, pH 8.0, lysed with 1% SDS, and incubated for 10 minutes at 37 °C. Samples were reduced with 100 mM DTT and analyzed by immunoblot after SDS-PAGE on 13% polyacrylamide gels with an antibody against the carboxyl-terminus of TM as described previously [Pelleted virus was resuspended in a small volume of HN buffer supplemented with 1 mM CaCleviously .To study the effects of the R99 peptide inhibitor of ALV-A infection on trimer formation, R99 (FNLSDHSESIQKKFQLMKEHVNKIG) and R992XP (FNLSDHSESPQKKPQLMKEHVNKIG) were synthesized to >95% purity by Research Genetics (Invitrogen) with N-terminal acetylation and C-terminal amidation. Liposomes were created from a mixture of a 1:1:1:1.5 molar ratio of phoshatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol. Lipids were obtained from Avanti Polar Lipids , mixed in chloroform, dried under argon, and lyophilized in glass tubes. The mixture was then rehydrated with HN buffer by shaking for 1 hour, sonicated in a water bath sonicator for 10 min, and extruded 25 times with an Avanti mini-extruder using a 100 nm pore size filter. Virus was mixed with liposomes with or without 100 nM sTva on ice. The R99 and R992XP peptides were dissolved in HN buffer, added to the virus/liposome/sTva mixtures to a final concentration of 100 μM, and incubated at pH 8 for 20 min at 37 °C. Samples were then adjusted to the indicated pH and incubated for 30 min at 37°C prior to neutralization and analysis as described above.To study the integrity of the SU-TM disulfide bond, samples were incubated with or with 100 nM sTva at pH 8.0 for 20 min on ice and then for 30 min at 37 °C at pH 5.0 or pH 7.4. Samples were neutralized, exposed to 100 mM DTT or buffer, and incubated at RT or 100 °C for 5 min prior to SDS-PAGE and analysis by immunoblot for TM. Immunoblots were then stripped with SDS/DTT and probed for SU using the mc8C5 mAb.To study the formation of the TM trimer induced by PMB, virus was incubated with or without the indicated concentrations of sTva for 20 min on ice at pH 8.0. Samples were then exposed to 1 mM PMB or buffer at the indicated pH for 30 min at 37 °C. Samples were neutralized and analyzed by immunoblot for TM as above.Virus was concentrated from supernatants by two cycles of ultracentrifugation through a 15% sucrose cushion in HN buffer, resuspended in a small volume of HN for 1 h at 4 °C, and incubated with 1.5 μM sTva to allow binding. PMB or SNB were dissolved in HN buffer and diluted into the virus/Tva samples to a final concentration of 1 mM. After incubation for 30 min at 37 °C, samples were quenched with a molar excess of cysteine-HCl (PMB) or glycine (SNB). Samples were lysed with NP-40 and immunoprecipitated with the antibody against the C-terminus of TM using Protein A beads (Pierce). Immunoprecipitated proteins were eluted from the beads by boiling in gel loading dye containing DTT, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed for biotin with streptavidin-HRP.Concentrated ALV-A was thawed on ice, and sTva was added to 5 μM. After incubation for 20 min on ice, NEM was added to 10 mM and incubated for 1 h at 37 °C. The sample was then concentrated by trichloroacetic acid precipitation and washed twice with cold acetone. The pellet was dried, re-suspended in SDS-PAGE loading dye with DTT, resolved by SDS-PAGE, and transferred to PVDF. The PVDF fragment containing the glycosylated SU was excised from the membrane, dried, and then boiled for 10 min in denaturing buffer (0.5% SDS with 1% β-mercaptoethanol). PNGase F (New England Biolabs) was added and incubated for 1 h at 37 °C. The deglycosylated sample was resolved by a second round of SDS-PAGE, quantified by SYPRO Ruby (Invitrogen) stain against a standard curve, excised from the gel, and submitted for analysis. The SU sample was reduced, alkylated, and digested with protease at the Harvard Microchemistry Facility. Samples were analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry on a Thermo Finnigan LCQ DECA XP Plus quadropole ion trap mass spectrometer.Amino acid substitutions in ALV-A Env were generated by PCR mutagenesis using overlapping primers. For each amino acid substitution, at least two base pairs of the codon were changed to minimize the chance of reversion. Initial PCR products were subcloned into a shuttle vector comprised of a Kpn I/Sal I restriction fragment of RCASBP(A)-EGFP in the pAB6-Env A vector, and mutagenesis was confirmed by sequencing. This restriction fragment comprises the entire Env ORF including some 5′ UTR sequence. A Kpn I/Stu I restriction fragment from the shuttle vector was then cloned into the RCASBP(A)-EGFP plasmid, and the presence of the mutation was again confirmed by sequencing. Mutant RCASBP(A)-EGFP plasmids were transfected into DF-1 cells with Superfect (Qiagen) according to the manufacturer's instructions to generate virus. Transfection was confirmed by EGFP expression. Replication-competent viruses were identified by monitoring the spread of acquired EGFP expression in the transfected cultures. Single cell clones of cells expressing noninfectious virus were produced by co-transfecting the RCASBP(A)-EGFP plasmid bearing the mutation with pBABE-puro, a puromycin expression plasmid. Cells were selected in puromycin (1 μg/ml), and colonies expressing EGFP were isolated and expanded. EGFP-positive clones were periodically tested to confirm the noninfectious phenotype by co-culture with uninfected DF-1 cells or by analyzing DF-1 cells exposed to filtered supernatant for acquired EGFP expression. The presence of the mutation was again confirmed by PCR and sequencing of the provirus in the transfected cells. For replication-competent mutant viruses, the mutation was confirmed in cells infected with filtered virus from the initially transfected cells to verify that the mutation could be stably transmitted.Virus titers in the supernatant of cells producing mutant viruses were determined by endpoint dilution on DF-1 cells. Env incorporation of mutant virus purified from supernatant by centrifugation (as above) was determined by SDS-PAGE and immunoblot using the antibody against the carboxyl-terminus of TM.The SU-A IgG expression construct was a gift from J. A. T. Young  . The CysTo determine the binding of SU-A IgG to Tva, 293 (negative control) or 293-Tva cells were incubated with increasing concentrations of SU-A IgG or Cys38Ser SU-A IgG for 1 hour on ice in PBS/1% FBS. Cells were washed twice with PBS/1% FBS and incubated with an Oregon green 488-conjugated goat anti-rabbit IgG (Invitrogen) for 1 hr on ice. Cells were washed twice and analyzed by flow cytometry.g (ave) using a TLA 100.1 rotor (Beckman Coulter). Fractions were collected from the top of the gradient, resolved by reducing SDS-PAGE, and analyzed by immunoblot using antisera against Rous sarcoma virus Prague C.Liposomes were created as described above. Pelleted virus was mixed with liposomes with or without 100 nM sTva and incubated for 45 min on ice. The R99 peptide was dissolved in HN buffer, added to the virus/liposome/sTva mixtures to a final concentration of 100 μM, and incubated at pH 8 for 30 min at 37 °C. Samples were then adjusted to the indicated pH, incubated for 30 min at 37 °C, and neutralized with 1 M Hepes. Neutralized samples were mixed with an equal volume of 80% sucrose in HNC, transferred to an ultracentrifuge tube (170 μl sample volume), and overlaid with 30% sucrose (340 μl) and 15% sucrose (100 μl) in HNC. Samples were centrifuged at 4°C for 1 hr at 179,000 x Cultured monolayers of DF-1 or 293-Tva cells were stained with 0.8 μg/ml R18 in growth media for 1 hr at 37 °C. Labeled cells were washed with PBS, and DF-1 cells expressing Cys38Ser Env or wild type RCASBP(A)-EGFP were harvested with versene, co-plated with the R18 labeled cells, and incubated for 2 h at 37 °C to form monolayers. The cells were washed twice with PBS, and then incubated with PBS at the indicated pH values for 15 min at 37 °C. Cells were then washed twice with PBS and returned to normal growth media. After an additional 30 min to 1 h incubation at 37 °C, images were taken, and syncytia were identified as large, multi-nucleated cells with diffuse EGFP fluorescence and R18 positive membranes. Parallel samples were harvested with versene and analyzed by flow cytometry for EGFP and R18.


Caenorhabditis elegans makes a developmental decision based on environmental conditions: larvae either arrest as dauer larva, or continue development into reproductive adults. There is natural variation among C. elegans lines in the sensitivity of this decision to environmental conditions; that is, there is variation in the phenotypic plasticity of dauer larva development. We hypothesised that these differences may be transcriptionally controlled in early stage larvae. We investigated this by microarray analysis of different C. elegans lines under different environmental conditions, specifically the presence and absence of dauer larva-inducing pheromone.The free-living nematode C. elegans lines under the same environmental conditions. The expression of approximately 2,000 genes differed between genetically different lines, with each line showing a largely line-specific transcriptional profile. The expression of genes that are markers of larval moulting suggested that the lines may be developing at different rates. The expression of a total of 89 genes was putatively affected by dauer larva or non-dauer larva-inducing conditions. Among the upstream regions of these genes there was an over-representation of DAF-16-binding motifs.There were substantial transcriptional differences between four C. elegans had substantial transcriptional differences. This variation may be due to differences in the developmental rates of the lines. Different environmental conditions had a rather smaller effect on transcription. The preponderance of DAF-16-binding motifs upstream of these genes was consistent with these genes playing a key role in the decision between development into dauer or into non-dauer larvae. There was little overlap between the genes whose expression was affected by environmental conditions and previously identified loci involved in the plasticity of dauer larva development.Under the same environmental conditions genetically different lines of  Caenorhabditis elegans makes a developmental decision between different larval fates. This decision is based on the 'suitability' of the environment for growth and reproduction. Under 'favourable' conditions, second stage larvae (L2) develop via two larval stages  into reproductive adults [via the L4 stage. Thus, the decision whether to develop into dauer larvae or into 'normal', non-dauer larvae is environmentally determined. The dauer or non-dauer developmental programme will, at least in part, be executed by transcriptional control.Developmental decisions and processes can be controlled transcriptionally. The free-living nematode e adults ,2. Howev stages L, L4 into stages L, L4 intoe adults , showingi.e. a high conspecific population density). Conversely, conditions that favour the development of non-dauer larva development are a high concentration of food and a low concentration of dauer pheromone. Higher temperatures favour the development of dauer larvae [The features of the environment that are used by larvae making this developmental decision are the concentration of food, the concentration of dauer pheromone and temperature. Dauer pheromone is a cue produced by all worms that acts as a measure of con-specific population density  and apper larvae .daf-16, have been shown to be key in the generation of these transcriptional differences [There has been extensive investigation into the genetic and molecular genetic control of the development of dauer larvae, which is known to be controlled by a TGF-β-like pathway, an insulin-like pathway and a guanyl cyclase pathway ,2. Thereferences -14. HoweC. elegans is still poorly understood. However, individual C. elegans are most often isolated from the wild as dauer larvae, rather than as reproducing adults [C. elegans and, secondly, that dauer larvae and the developmental decision whether or not develop into dauer larvae is likely to be under strong natural selection. Previously we have compared the plasticity of dauer larva development of different lines of C. elegans [C. elegans differ in their plasticity of dauer larva development [i.e. low plasticity lines); in other lines, the proportion of individuals that develop as dauer larvae will increase rapidly with the concentration of dauer pheromone (i.e. high plasticity lines).The natural history of g adults . This ob elegans ,17. Plaselopment ,17. For C. elegans is due to inter-line transcriptional differences. To investigate this we have investigated the transcriptional profiles of C. elegans lines with different phenotypic plasticities of dauer larva development. We further hypothesised that the different dauer larva development plasticities were most likely to be due to interline differences in the 'decision' and early initiation of development into non-dauer or dauer larvae. For this reason we compared the transcriptional profile of early stage larvae exposed to dauer larva or 'normal', non-dauer larva-inducing conditions.Genetic analysis of variation in the plasticity of dauer larvae formation has identified a number of quantitative trait loci (QTL) that control it . Given tC. elegans lines N2 and DR1350 and two recombinant inbred lines (RILs), RIL-14 and RIL-17 were used. N2 and DR1350 were obtained from the Caenorhabditis Genetics Center. The RILs were previously generated by taking F1 progeny of a N2 × DR1350 cross which were then allowed to self-fertilise for at least 30 generations, as previously described [Escherichia coli OP50 food source.escribed ,17. All escribed  with an Previously, the phenotypic plasticity of dauer larva development of these lines was determined ,17,19. NC. elegans whole genome microarrays.These analyses were undertaken in two experiments. In experiment one N2 and DR1350 were analysed; in experiment two RIL-14 and RIL-17 were analysed. For each of the four lines, L1s were grown in 'normal', non-dauer larva-inducing or in dauer larva-inducing conditions until they were halfway through the L2 stage, at which point the larvae were harvested for the preparation of RNA for use with E. coli OP50 food source was then added to achieve a final concentration of 20 mg/mL; this was the 'normal', non-dauer larva-inducing conditions. We had previously determined that this concentration of food was sufficient for L1s to grow to adulthood and, thus, that these are 'normal' and non-starvation conditions (data not shown). The dauer larva-inducing conditions were the same, but with the addition of 450 μL of dauer pheromone to each tube. All cultures were maintained for a further 8.5 hours, at 25°C while being shaken at 200 rpm; at this time the larvae are temporally half-way through the L2 stage. Development was confirmed by microscopic analysis of the larvae in pilot experiments (data not shown). At this point 0.5 mL of each culture was removed and maintained in the same conditions for a further two days to confirm the phenotype  of the worms grown under these two conditions. The remainder of each sample was centrifuged for two minutes at 850 g, the supernatant removed and the worms resuspended in M9 buffer [g for 6 minutes at 4°C on a 60% v/v: 40% v/v Percoll gradient in M9 to remove residual E. coli OP50, the worms removed from the interface, washed three further times by sedimentation and resuspension in M9, before being finally resuspended in a small volume of M9 to which an equal volume of Tri reagent (Sigma-Aldrich) was added. Samples were then snap frozen in liquid nitrogen and stored at -80°C. RNA was extracted following the manufacturer's instructions. For each line of C. elegans there were three biological replicates for each of the two environmental conditions. Dauer pheromone was prepared from liquid cultures of N2 as previously described [E. coli OP50 food source was prepared from shaken liquid cultures grown overnight in LB media at 37°C, after which the bacterial suspension was centrifuged for 15 minutes at 5,500 g, and the supernatant removed, with the pellet of bacteria resuspended in M9 at a concentration of 0.2 g/mL.For each replicate of each line, 30,000 age synchronous eggs were liberated from hypochlorite treated gravid hermaphrodites  that had9 buffer ; this waC. elegans GeneChip genome arrays (Affymetrix) following the manufacturer's instructions. These chips represent 22,500 transcripts of the expressed C. elegans genome based on the December 2005 genome sequence and GenBank release 121. The microarray data reported here have been deposited at ArrayExpress http://www.ebi.ac.uk/microarray-as/aer/entry with accession number E-MEXP-1808 and E-MEXP-1810 and have been submitted to WormBase http://www.wormbase.org.RNA samples for microarrays were processed and microarray hybridisation was performed by the Gene Microarray centre of the Institute of Child Health, University College London. RNA integrity was checked using a Bioanalyser 2100 (Agilent). 5 μg of total RNA from each sample was converted to cDNA using an oligo(dT) primer and a microarray cDNA synthesis kit (Roche). The cDNA was then labelled using an Affymetrix labelling kit and the product fragmented and hybridised to 2 transformed intensities for individual features were scaled by subtracting the mean chip log2 intensity value, and centred by dividing by the chip standard deviation. Each experiment was analysed separately, with between-chip normalisation and distribution normalisation applied across all chip data within that experiment as described elsewhere [INE , TREATMENT (non-dauer vs dauer larva-inducing) and the LINE × TREATMENT interaction, using a published PERL script [F1,8 values  for the effects of LINE, TREATMENT and LINE × TREATMENT for each gene were determined.Data were analysed separately using a series of custom PERL scripts. For within-chip normalisation, the loglsewhere . BrieflyL script . In thisTo validate the microarray results we used semi-quantitative RT-PCR to analyse the expression of 15 genes in N2 and DR1350. To do this 0.5 μg of total RNA was transcribed into cDNA and this template used in PCR reactions, all as previously described  for 30 cth September 2007) [C. elegans gene expression topomap. This determined the probability that the occurrence of the selected 'query' genes within each of the 'target' groups of genes was random. To identify potential regulatory regions in sequences upstream of genes of interest, 500 bp of sequence immediately 5' of each gene (WormBase release WS180) was analysed using BioProspector [C. elegans genes. We used a hypergeometric test to determine the probability that the observed distribution of such regions among genes of interest was different to the distribution among all genes.Gene annotation, including gene ontology (GO) annotation was taken from WormBase release WS180 (7er 2007) . Genes oer 2007) , to exisospector , with deAnalysis and quality control of the microarray data showed that data were available for all replicates and treatments for 15,792 genes from experiment one (N2 and DR1350) and for 14,759 genes from experiment two (RIL-14 and RIL-17). Of the 15 genes analysed by semi-quantitative PCR, 13 could be successfully amplified and the expression of 9 (69%) was concordant with the microarray data .C. elegans lines within the same environmental conditions. There were 2,920 genes whose expression differed significantly (LINEp < 0.001) between N2 and DR1350  and 221 whose expression differed significantly (LINEp < 0.001) between RIL-14 and RIL-17 . These genes are listed in additional file C. elegans lines across all developmental stages also found substantial inter-line transcriptional differences [Saccharomyces cerevisiae has shown that a naturally occurring single frameshift mutation controls 45% (103 genes) of the transcriptional differences between two lines [S. cerevisiae have shown inter-line differences in gene expression are controlled by trans-acting loci, that are not necessarily transcription factors [i.e. 'noise') which is genetically controlled [There were substantial differences in gene expression between the different ferences . Analysiwo lines . Thus, s factors . Furtherntrolled .etc. compared with N2. Analogously, in N2 there is significantly greater expression of mount 6 and 2 genes etc., compared with DR1350. RIL-14 and RIL-17 also appear to have different transcriptional profiles (Table e.g. during the L1 stage), but their transcriptional effect is amplified as development proceeds.We compared the occurrence of genes significantly differently expressed in N2 and DR1350 to previously prepared gene lists . This shC. elegans lines has not resulted in the generation of erroneous data.There are significant genome sequence differences between N2 and DR1350  which maC. elegans lines are following different transcriptional profiles. An alternative possibility is that they are actually following the same transcriptional profile, but that the lines are moving through this at different rates. In support of this, for example, DR1350 reaches sexual maturity approximately 2–3 hours earlier than N2 at 25°C [The analyses above suggest that these  at 25°C , suggestmlt-8, 9, 11, sqt-1, 3, dpy-7, 9 (p < 0.001); daf-9, mlt-10 (p < 0.05)) was significantly greater in DR1350 compared with N2; one was not . This suggests that DR1350 is more developmentally advanced (i.e. closer to the moult into L3) compared with N2. Similarly, in RIL-14 and RIL-17 the expression of 6 of these genes ; mlt-10, sqt-1, dpy-7, 9 (p < 0.05)) were significantly greater in RIL-14 compared with RIL-17; four were not ). Together, these data strongly suggest that DR1350 and RIL-14 are more developmentally advanced compared with N2 and RIL-17, respectively, and therefore that the interline differences in transcription that we have observed may be due to differences in developmental progression. The changes in gene expression over the C. elegans life-cycle are therefore likely to be even more complex than those identified so far [To investigate this further we compared the expression of 10 genes that are key indicators of the early stages of the preparation for the L2 – L3 moult. These 10 genes are known to be involved in the moulting process and there is an increase in the abundance of their transcripts from the mid-L2 stage -35. The d so far .It is notable that DR1350 and RIL-14 have lower phenotypic plasticities of dauer larva formation compared with N2 and RIL-17. It is therefore possible that the lines have different sensitivity to dauer larva-inducing conditions . If the REATMENTp < 0.001) in their expression between these two conditions. 13 genes were expressed comparatively more in dauer larva-inducing conditions, 16 in non-dauer larva-inducing conditions. In RIL-14 and RIL-17, 65 genes differed significantly (TREATMENTp < 0.001) in their expression between these two conditions; 22 genes were expressed comparatively more in dauer larva-inducing conditions, 43 in non-dauer larva-inducing conditions. The expression of 10 genes was affected by environmental conditions differently between N2 and DR1350 (i.e. a genotype-by-environment effect) (ISOLATE × TREATMENTp < 0.001), with the expression of 4 of these also differing between treatments (TREATMENTp < 0.001), but there was no such effect for RIL-14 and RIL-17  effects . Our finREATMENT and LINE × TREATMENTp < 0.001) in larvae exposed to non-dauer larva or dauer larva-inducing conditions in at least one of the experiments   [Analysis of these 89 genes showed that those up-regulated in dauer larva-inducing conditions were over-represented in mount 8 Table . No speclopment) ,10. LarvC. elegans genome the CTTATCA sequence occurred within the same region in c.5% of genes. Therefore, this sequence is significantly over represented (p < 0.0001) among the putative regulatory regions of the 89 genes identified here. This sequence was previously identified as being over-represented upstream of genes whose expression differs between daf-2(RNAi) (wild-type lifespan) and daf-16(RNAi); daf-2(RNAi) (long lifespan) worms [daf-2(RNAi) (wild-type lifespan) and daf-16(RNAi); daf-2(RNAi) (long lifespan) worms [daf-16 [via the action of DAF-16.Among these 89 genes Figure , Table 3n) worms . It has n) worms . This sen) worms . Furthern) worms , is presn) worms , was fou [daf-16 . Overallc. 200 kbp region on chromosome II; this QTL does not encompass any loci previously identified to control dauer larva development [Previously we have identified QTLs that control dauer larva formation under different environmental conditions and the plasticity of dauer larva development in N2 and DR1350 . One of elopment . Of the trans-acting loci. These trans-acting loci may be among the many loci that are differently transcribed between the lines.Overall, comparison of the QTL and expression analyses of the plasticity of dauer larva development show poor correspondence. This therefore suggests that these dauer larva development traits are genetically complex controlled, at least in part, by C. elegans lines in the transcriptional profile of mid-second stage larvae. These different profiles are commensurate with the likely other genomic differences between C. elegans lines. These transcriptional differences are concentrated in groups of putatively functionally-related genes, suggesting that each of these lines is following a different transcriptional profile. However, the gene expression data also suggest that these lines differ in their developmental rate. Therefore the apparent different transcriptional profiles, may also be a consequence of this different timing. Further, the different phenotypic plasticities of the lines may also be explained be different developmental rates, and hence inter-line differences in the 'window of sensitivity' to environmental change, of the lines.We have found that there are substantial differences between C. elegans lines. Comparison of these gene expression data with previous QTL data, reinforce that this phenotypic plasticity trait is genetically complex.We identified a small number of genes whose expression was altered in dauer or non-dauer larva-inducing conditions. Changed expression of so few genes is compatible with these early stage larvae commencing development as dauer or non-dauer larvae. We found evidence for a DAF-16 binding sequence upstream of many of these genes, suggesting that DAF-16 may co-ordinately control these changes in gene expression. These data, though, do not obviously expose the bases of the different phenotypic plasticities of dauer larva development among these four SCH and MEV generated the RILs, AS prepared the larvae and RNA, GLAB analysed the hybridisation data with AS and SCH, SCH undertook further analysis and co-wrote the manuscript with MEV. MEV conceived and directed the study. All of the authors read and approved the final manuscript.Inter-line differences in gene expression. The genes whose expression is significantly affected by LINE (p < 0.001), in experiment one (N2 and DR1350) and experiment two (RIL-14 and RIL-17) showing the Affymetrix identifier and annotation, the ANOVA p values for LINE, TREATMENT and LINE × TREATMENT, effects and the mean hybridisation intensities for DR1350 and N2 and for RIL-14 and RIL1-7, under non-dauer and dauer-inducing conditions and the direction of the N2 and DR1350 and the RIL-14 and RIL-17 difference.Click here for file


Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually  and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA. Mammalian ribosomal RNA genes are comprised of several hundreds of transcription units clustered on a number of chromosomal loci Polymorphic variations in rDNA are well documented Xenopus oogenesis, in which the rDNA array was amplified several thousand-fold to boost rRNA synthesis Another not-well-explored area in the regulation of rRNA synthesis is its cell-type-specificity Bnc1, MGI) is a zinc finger protein with highly restricted cell-type distribution–it is mainly expressed in keratinocytes and the reproductive germ cells. BNC1 interacts with rDNA promoter and influences Pol I transcription  and the surrounding 6 kb sequence Fig. 1D.To assess the size of the rDNA pool detected by our Southern analysis, we measured the copy numbers of the Stu I and Sty I RFLPs using a set of markers  of the estimated copies of the rDNA pool studied here, i.e., individually, for I+II, 29% (8/28); III, 33% (4/12); IV, 33% (4/12); V, 50% (2/4); VI, 44% (4/9) and VII, 50% (4/8) )(Genbank accession numbers are given in Within the promoter region (+1 to −300) of the seven v-rDNAs, only two nucleotide substitutions were present Fig. 4A,Each of the seven v-rDNA promoters contained three HpaII/MspI sites , which were used to assess the DNA methylation status of the promoters Fig. 2C.Multi-sequence alignment showed variant-specific SNPs in the transcribed region of v-rDNA Fig. 3B.We used the variant-specific PCR assay to assess the expression level of each v-rDNA Fig. 6A.Using quantitative PCR (qPCR), we investigated the relative transcript level of each v-rDNA in brain, skin, and testis Fig. 7A.21.7 or 3.7×106-fold difference. The same assay showed that the Ct value of genomic v-rDNA IV was 25.3±2.1 and that of v-rDNA V 27.1±2.4, an approximately 4-fold difference (21.8 = 3.48). The ratio of Ct values of RNA and DNA suggested that for each copy of v-rDNA IV there were 128 transcripts , whereas no transcript was detected for v-rDNA V. This result demonstrated that the variations in expression level could not be explained by copy number alone, and thus differential transcription modulation or post-transcription processing should be considered.We also investigated the relationship between expression level and gene copy number of variants IV and V in the testes of 129Sv/C57BL-6J hybrid mice. Variant IV was highly expressed whereas V was virtually silent. Furthermore, their PCR assays had similar amplification efficiency Fig. 7C.Based on the data presented here, we propose that mouse rDNA array contains several variant types. The expression of each variant appears to be regulated independently from others and for some variants , expression is tissue-specific. We describe several lines of evidence to support this proposal. Guided by the stable rDNA RFLPs, we cloned seven v-rDNAs; copy number analyses suggest that most variants contain between 8–15 copies; we identified variant-specific SNPs in the transcribed sequence and variant-specific PCR revealed variable expression of these v-rDNAs in different tissues; and v-rDNA expression profiles are conserved within and among mouse strains.Our results are consistent with previous reports on the v-rDNA RFLPs. A previous study used two restriction enzymes (Eco RI and Hind III) to examine six inbred CBA/H-T6 individuals and found no variation in their rDNA RFLPs The DNA methylation status of the v-rDNA also suggests that we are dealing with a subset of rDNA. DNA methylation measurements by HpaII sensitivity as well as by bisulfite sequencing showed that the promoters of the seven v-rDNAs are hypomethylated, whereas the same method detected hypermethylated rDNA promoters in the genome. Furthermore, bisulfite sequencing suggested that ∼17% of the mouse genomic rDNA promoters are hypomethylated . On the other hand, if the methylated rDNAs are in a different configuration or their restriction fragment length varies from unit to unit, then restriction digestion will produce a smear of bands on the gel, which would not be identified and cloned by our method. A recent study of human rDNA array did show rDNA units of variable length, as well as existing in configurations other than tandem repeats  The copy number analysis also suggests that each type of v-rDNA contains between 8–15 copies, which are arranged in tandem . An ambiguity is that we could not measure by Southern blot the copy number of v-rDNA IV and V separately. However, qPCR measurements suggest that their copy number ratio is 3.48 Fig. 7C.The expression studies suggest that the v-rDNA types are regulated differentially. The constitutively expressed v-rDNAs  represented approximately 56% of the copies of the v-rDNA detected. The selectively expressed III and VI constitute about 29% copies, in which VI is expressed more widely than III, suggesting that they are individually regulated. The silent V and VII have ∼15% of the copies. Our data show that the three v-rDNA regulatory groups are present in six mice of three strains, which strongly suggest that their regulation is not randomly determined. It should be noted that our analysis employs tissues containing many cell types, each of which may have a unique profile of v-rDNA expression. When assayed as a mixture, the unique v-rDNA profile of each cell type may be obscured . Furthermore, our observation is limited developmentally to a narrow window in adulthood. Therefore, our results are likely an underestimate of the complexity of v-rDNA regulation. This point is of particular relevance in interpreting the apparent lack of expression of v-rDNA V and VII. It remains possible that these v-rDNAs are expressed in a small number of cells or at a particular developmental stage, and thus not detected by our screen. Consistent with this proposal is that v-rDNA VII is expressed in corneal epithelial keratinocytes Although our data do not discern how the regulation is achieved  for the actively transcribed rDNA , a number of transcriptional and processing regulatory mechanisms have been reported  following manufacturer's instructions. Sequence alignment was first performed by the computer program Clustal-W implemented in Vector NTI (Invitrogen) or as a web-based program  Table S1Genbank accession numbers of v-rDNA sequences(0.02 MB DOC)Click here for additional data file.Methods S1(3.67 MB PDF)Click here for additional data file.


Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B. pertussis infection than C3H mice, which could partially be ascribed to the B. pertussis susceptibility locus-1 (Bps1) on chromosome 12. The presence of C57BL/10 genome on this locus instead of C3H genome resulted in a decreased number of bacteria in the lung. To further elucidate the role of host genetic factors, in particular in the Bps1 locus, in B. pertussis infection, and to identify candidate genes within in this region, we compared expression profiles in the lungs of the C3H and HcB-28 mouse strains following B. pertussis inoculation. Twelve and a half percent of the genomes of these mice are from a different genetic background.Susceptibility to B. pertussis inoculation 2,353 genes were differentially expressed in the lungs of both mouse strains. Two hundred and six genes were differentially expressed between the two mouse strains, but, remarkably, none of these were up- or down-regulated upon B. pertussis infection. Of these 206 genes, 17 were located in the Bps1 region. Eight of these genes, which showed a strong difference in gene expression between the two mouse strains, map to the immunoglobulin heavy chain complex (Igh).Upon B. pertussis infection are highly identical between the two mouse strains despite the differences in the course of B. pertussis infection. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains. Genes in the Igh complex, among which Igh-1a/b, are likely candidates to explain differences in susceptibility to B. pertussis. Thus, by microarray analysis we significantly reduced the number of candidate susceptibility genes within the Bps1 locus. Further work should establish the role of the Igh complex in B. pertussis infection.Gene expression changes upon  Bordetella pertussis is a gram-negative bacterium that can cause the respiratory disease known as pertussis or whooping cough in humans. Susceptibility to this disease and its course vary widely between individuals [B. pertussis infection, underlining that infection is influenced by host genetic factors[B. pertussis infection has been indicated, such as the toll-like receptor 4 (Tlr4) gene [B. pertussis susceptibility locus 1, 2, and 3  [ividuals . We havec factors,3. In adr4) gene -5, the ir4) gene , and thrB. pertussis infection [Bps1, 2, and 3. Especially Bps1 displayed strong linkage with susceptibility to B. pertussis infection. The Bps1 locus is located on chromosome 12, spanning a region of 185 genes, of which one or more genes have a dominant positive effect on the clearance of B. pertussis in the lung, and/or the reduction of bacterial multiplication. However, the mechanism by which genes within this region influence the course of infection is not clear. Two other loci, Bps-2 and Bps-3, showed genetic interaction and are located on chromosomes 5 and 11, respectively [We have used recombinant congenic mouse strains (RCS) as a tool to facilitate the mapping of low-penetrance quantitative trait loci that control complex traits such as a nfection . RCS arenfection . HcB/Demnfection ,10. HcB-Bps-1 has not yet been validated, we believe that the significance of this locus warrants further study. To further elucidate the role of host genetic factors, in particular the Bps1 locus, in B. pertussis infection, and to identify candidate genes within this region, we studied expression profiles in the lungs of mice following B. pertussis inoculation. The traditional approach for mapping genes in susceptibility loci is a combination of positional cloning and linkage analysis [B. pertussis infection, and we especially focused on differentially expressed genes in the lungs of infected and non-infected mice located in Bps1,-2, and -3 [B. pertussis infection. These genes are involved in various immune-related processes, such as the acute-phase response, antigen presentation, cytokine production, inflammation, and apoptosis. Nine of the differentially expressed genes are located in Bps1, 13 are located in Bps-2, and 62 are located in Bps-3.Although analysis ,12. Althanalysis ,13, the , and -3 . We founB. pertussis infection, in order to further identify candidate susceptibility genes without the need for positional cloning. We hypothesized that the phenotypic differences displayed by these mice in the response to B. pertussis can partly be explained by a different gene expression profile between the mouse strains, and that this approach could lead to the identification of candidate genes affecting the course of infection. Using this approach we reduced the number of candidate susceptibility genes within the Bps1 locus.In the present study we compared the gene expression profiles in the lungs of two mouse strains, i.e. HcB-28/Dem and C3H, which showed a different course of 7 colony forming units (CFU) of the streptomycin-resistant Tohama strain of B. pertussis (B213) in 40 μl Verwey medium , or as a control with Verwey Medium only . One, three, and five days after inoculation, 8 infected and 8 control mice were euthanized. To remove blood from the lungs, mice were perfused with phosphate-buffered saline . Subsequently, the lungs and trachea were collected [Forty-eight female HcB-28/Dem and 48 female C3H/DISnA (the background strain of the HcB-28/Dem) mice were intranasally inoculated with 2*10ollected ,16. For B. pertussis bacteria was determined in the trachea to confirm a proper infection [The number of viable nfection  .All For the analysis of intrinsic immunological and cellular differences between the two mouse strains, independent from infection, blood and spleen samples were collected from five untreated female mice of both strains.Breeding pairs of C3H/DISnA and HcB-28/Dem were supplied by the Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York. Female mice were bred to the appropriate numbers at our animal testing facility in Bilthoven, the Netherlands. Mice were acclimatized for at least one week before the start of the experiments. Mice received standard laboratory food  and tap water ad libitum. All animal experiments were approved by the Institute's Animal Ethics Committee and were performed according to NIH guidelines  and DutcMice were weighed before inoculation, and subsequently every day after inoculation to determine the relative change in weight. Lung weights were determined post mortem as quantitative parameter for lung inflammation. Formalin-fixed lungs were embedded in paraplast . Sections (5 μm) were stained with hematoxylin-eosin. Lung lesions were examined for infiltration of inflammatory cells in the peribronchiolar space (peribronchiolitis), infiltration of inflammatory cells in the alveoli , infiltration of inflammatory cells in the perivascular space (perivasculitis), hypertrophy of mucus-producing glands, free protein (exudate), and eosinophilia. Lung lesions were scored semi-quantitatively as absent, minimal, slight, moderate, marked, or strong, as previously described .Spleens were harvested and weighed. Cell suspensions were prepared and the number of nucleated cells per spleen was determined using a Coulter Counter Z2 . The percentage of B- and T-cells, as well as T-cell subsets were determined using a fluorescence-activated cell sorter . T-cells were detected with phycoerythrin-labeled (PE) anti-CD3ε antibodies . CD4+ T-cells were determined using allophycocyanin-labeled (APC) antibodies (Molecular probes) and CD8+T-cells were determined using fluorescein isothiocyanate-labeled (FITC) antibodies (Molecular probes). B-cells were determined using PE-labeled anti-CD19 antibodies (Molecular probes).3H-thymidine uptake. The cells were stimulated by adding 10 μg/ml Concavalin A , 70 μg/ml lipopolysacharide  or 15 μg/ml Lectin (B- and T-cell stimulus) to the culture medium. Cells were incubated for 72 hr at 37°C. 3H-thymidine was present during the final 24 hr of culture. See [The cell suspensions were used to measure the spontaneous (medium only) or stimulus-induced ure. See  for detaThe morphologic hemogram was determined using an ADVIA 120 Hematology system .Blood was collected in Vacuette Minicollect tubes , and serum was removed by centrifugation. Immunoglobulins were determined using the multiplex Beadlyte Mouse Immunoglobulin Isotyping Kit  for the Luminex platform , as prescribed by the manufacturer.Microarray analysis experiments were performed as described previously . BrieflyBps1 region spotted in duplicate , as well as appropriate controls and blank spots. The Bps-1 oligonucleotides were annotated according to NCBI mouse genome build 34.1; all other oligonucleotides were annotated according to the Sigma-Compugen Mouse oligonucleotide library.Microarray slides were spotted at the Microarray Department of the University of Amsterdam. The slides contain 21,997 65-mer oligonucleotides from the Sigma-Compugen Mouse oligonucleotide library, 192 additional 65-mer oligonucleotides of genes of which 185 are located in the P-value for enrichment.At least 5 slides per group were used. Raw microarray signal data were normalized and analyzed in the R statistical software environment -23. SignB. pertussis from the lungs of infected RCS mice, and established that HcB-28 mice contained lower numbers of bacteria in their lungs seven days post inoculation compared to C3H mice . The gene expression levels of all 2,559 genes are presented in additional file B. pertussis susceptibility.The three mock-inoculated groups of each mouse strain, euthanized at days one, three and five, were found to be statistically indistinguishable from each other, i.e. both within the same strain and between strains (i.e. there were no genes with FDR < 0.6). Of the approximately 22k genes tested, 2,559 genes were significantly differentially expressed either in the two mouse strains or upon B. pertussis infection followed a similar trend in time. One day post-inoculation differential gene expression was already observed, but the effect was stronger on days three and five post-inoculation. Thus there was no identifiable subset of genes that displayed a different kinetic trend in gene expression.The 206 genes that were differentially expressed between the two strains were all unaffected by infection, but displayed a difference in basal expression level. Of the 206 strain-dependent genes, 107 genes were expressed at a higher level in C3H/DISnA mice (group A) and 99 genes were expressed at a higher level in the HcB-28/Dem mice (group B). Of the 2,353 infection-regulated genes, 963 were down-regulated upon infection (group C), and 1,390 were up-regulated upon infection (group D). All genes that were affected by B. pertussis inoculation. The most important GO categories, i.e. with the highest percentage differentially expressed genes, are summarized in Table Bps1 (8 in group A and 9 in group B)[Bps1 are presented in Table BC022687, up to 3.7-fold higher in C3H mice) and for Immunoglobulin heavy chain 1 in group B . Eight genes within Bps1 (6 in group A and 2 in group B) showed a strong difference (i.e. at least two-fold) in gene expression between the two mouse strains, and map to the immunoglobulin heavy chain complex (Igh).To assign functions to the differentially expressed genes, we classified them according to Gene Ontology (GO) categories. In addition, we determined enrichment for location and biological functions to identify pathophysiological processes involved in the response to  group B). All theB. pertussis infection, 1,702 genes were identical to the genes we have described previously to be regulated upon B. pertussis infection [B. pertussis, most of which were weakly up- or down-regulated. Most up-regulated genes upon B. pertussis infection (group D) are involved in immune- and inflammation-related processes or in generic processes, while most down-regulated genes (group C) are involved in non-immune processes such as muscle contraction.Of the 2,353 genes that were differentially expressed after nfection . Thus byP = 0.0003) in the C3H/DISnA mice (1.1*1012/l) compared to the HcB-28/Dem mice (9.6*1011/l), and a slightly higher number of white blood cells (P = 0.006) in the C3H/DISnA mice (7.3*109/l) compared to the HcB-28/Dem mice (6.5*109/l), but no differences in the numbers of red blood cells, reticulocytes, lymphocytes, neutrophils, eosinophils, and basophils (data not shown).To investigate possible differences in the basal immunological parameters between the two strains, we analyzed hematologic parameters, mitogen-induced splenocyte proliferation, and lymphocyte subset distributions. Because the two mouse strains displayed clear differences in basal gene expression, we wanted to examine if these differences led to alterations in blood cell composition or basal immune status. We observed a slightly higher number of platelets (3H-thymidine uptake. There was no difference in proliferation between the two strains after ex vivo stimulation with Con A (T-cell stimulus), LPS (B-cell stimulus) or Lectin (B- and T-cell stimulus) (data not shown). We observed no differences in the number of CD19+ (B), CD3ε+ (T), CD4+(Th) and CD8+ (CTL) cells (data not shown).Splenocyte proliferation was determined by mitogen-induced B. pertussis-inoculated mice . C3H mice had significantly higher serum levels of IgM, IgG1, IgG2a and IgG3, while HcB-28 mice had significantly higher IgG2b levels compared to C3H mice. Importantly, HcB-28 mice had no detectable levels of IgG2a in the serum at all, which is consistent with the difference in gene expression at the Igh-1 locus between the two mouse strains.Finally we determined levels of IgM and subclasses of IgG in the sera of the mock- and e Figure . InfectiTo examine whether the two mouse strains differed in weight loss after infection, we determined their body weights after infection. Infected mice lost three to four percent of their body weight during the first five days post-inoculation, while all mock-inoculated mice gained weight during these five days Figure . No signB. pertussis-infected mice had relative lung weights of up to three percent. This difference in relative lung weights due to B. pertussis infection is significant from day one till day five post-inoculation, but was similar in both mouse strains (data not shown).As a quantitative endpoint for inflammation, we determined lung weights relative to body weight. All mock-inoculated mice had relative lung weights of approximately one percent five days after inoculation, while B. pertussis causes an acute inflammatory response that is characterized by influx of polymorphonuclear leukocytes (PMNs) and macrophages, starting in the perivascular and peribronchiolar areas on day one, and extending to alveolar walls and lumina on days three and five. No differences were observed in the histopathological lesions findings between the two mouse strains  in HcB-28 mice [Bps1 locus is located on chromosome 12, spanning a region of 185 genes and has a dominant positive effect on the clearance of B. pertussis from the lung. In this study we examined gene expression profiles in HcB-28 and C3H mice, which differ in their susceptibility to B. pertussis. Twelve and a half percent of the genomes of these mice are from a different genetic background [B. pertussis infection could (partly) be explained by a different gene expression profile between the mouse strains.Studying genetic differences in susceptibility to ckground ,10. The ckground ,12. Thisckground ,13, but B. pertussis infection in C3H mice induces a wide transcriptional response, which appears to be partly specific for B. pertussis and partly non-specific [B. pertussis inoculation, and most up-regulated genes are involved in immune- and inflammation-related processes or in generic processes, while most down-regulated genes are involved in non-immune processes. In the present study, we found that HcB-28 and C3H mice showed a similar gene expression profile upon infection and identified approximately 650 additional genes regulated by B. pertussis, most of which were weakly up- or down-regulated. However, the gene expression profiles and enrichment for GO categories were identical as described in the previous study. A substantial number of genes and pathways suggest a central role of PMN recruitment and activation in the pathogenesis of B. pertussis infection. The transcriptional profiles further indicate in particular the significance of TLR activation and apoptosis [B. pertussis infection were not detected in the present study. These genes were borderline significantly regulated (median FDR of 0.03) and only slightly induced (median 1.4-fold). These genes are therefore probably less important in the host response to B. pertussis infection. The finding that this list of 139 genes does not show significant enrichment for any GO-term, including immunological terms, corroborates this.We have previously shown that specific . This stpoptosis . The reaB. pertussis infection between the two mouse strains. The major difference is that HcB-28 mice did not have detectable IgG2a serum levels.Although the two mouse strains differ in 12.5% of their genome (12.5% of the genomes of these mice are from a different genetic background), we observed no marked differences in their phenotypical characteristics other than the previously observed difference in bacterial numbers in the lungs after infection Figure . C3H micB. pertussis-inoculated mice. The majority of these genes (65%) are unannotated. These unannotated genes tend to have no GO functional annotation, because they are not "regular" protein-coding genes and many of them are not (sufficiently) mapped to a chromosomal locus to warrant including them under chromosome 12 or Bps-1. The mentioned 23 genes, which were mapped to chromosome 12, are significant at P = 5.29e-014 . The same test applied to all other mouse chromosomes yielded P values > 0.05. Twenty-three out of the 206 genes were located on chromosome 12, which can be explained by the fact that approximately one fifth of the genetic variation between the two mouse strains is due to variation on chromosome 12 [B. pertussis infection. Alternatively, such genetic differences may be explained by genes that are not differentially regulated between these two strains of mice, or by processes at present not fully characterized and possibly involving differential expression of genes by mechanisms such as microRNA's. Remarkably, 17 of these genes were located in the Bps1 region, 8 of which mapped to the Igh complex. Among these 8 genes were the Igh-1 gene and genes that encode for Ig heavy chain variable regions. We observed a significantly higher expression (up to 2.8-fold) of the gene variant of Igh-1 isotype b in HcB mice compared to C3H mice. The Igh locus is genetically polymorphic and very complex [Igh-1 gene exists in 2 major genetic variants (Igh-1a and Igh-1b) with 83.8% similarity [Igh-1a allele codes for the heavy chain of IgG2a while the Igh-1b allele codes for the heavy chain of IgG2c [Igh-1b and are therefore incapable of producing IgG2a, while mouse strains such as BALB/c only contain the gene variant of Igh-1a and are therefore incapable of producing IgG2c [Igh-1 spotted on the microarray, was designed based on accession number XM_484178 annotated as Igh-1a. Because this annotation is based on the reference C57BL/6 mice, this oligo can be considered as Igh-1b annotated. In the sera of HcB mice we detected no IgG2a while C3H mice had significant titers of IgG2a, thereby confirming the expression results. It is tempting to speculate if and how genes within the Igh complex may affect differences early in the course of B. pertussis infection. Possible mechanisms may include differences in transcriptional gene regulation affecting immune responsiveness, different function of the IgG2a or c isotypes, or different usage of V chains. This latter possibility might imply the existence of "natural antibodies" reacting with B. pertussis epitopes. It has previously been shown that genes within the Igh-1 locus are predominantly associated with the course of a herpes simplex virus type-1 (HSV-1) infection in mice by an unknown mechanism [Igh-1 locus but could not simply explain the differences in HSV-1 susceptibility [Igh-linked genes have further been implicated in T suppressor cell activity [We observed 206 genes that were differentially expressed between the two mouse strains, but these genes were identically expressed in mock- or  complex . The Ighof IgG2c . Mouse sng IgG2c . The oliechanism -33. Pro-echanism . Interesactivity ,36.Bps1 locus by microarray analysis. Gene expression changes upon B. pertussis infection appear highly identical between C3H and HcB-28 mouse strains despite the different course of B. pertussis infection in these strains. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains or by processes other than differential gene expression. Genes in the Igh complex, among which Igh-1, may be likely candidates to explain differences in susceptibility to B. pertussis. Further work should establish the role of the Igh complex in B. pertussis infection and determine its mode of action.In this study we reduced the number of candidate susceptibility genes within the Raw data as well as the detailed description of the experiment was uploaded to the freely accessible online database ArrayExpress . ExpressThe author(s) declares that there are no competing interests.SB: carried out the infection and microarray studies and wrote the manuscript. RJV: participated in the study design and coordination and helped to draft the manuscript. JLAP: participated in the design of the microarray analysis and performed the statistical analysis. ERG: carried out the immunoassays. PWW: evaluated the lung pathology. TMB: participated in the design of the microarray analysis. PD: responsible for the genetic model of recombinant congenic mice. HJK, FRM, and BH: participated in the study design and coordination. TGK: conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Comparative gene expression profiling in two congenic strains of mice following Bordetella pertussis infection. The data present the expression levels of all 2,559 genes.Click here for file


Plant latex is the cytoplasm of highly specialized cells known as laticifers, and is thought to have a critical role in defense against herbivorous insects. Proteins abundantly accumulated in latex might therefore be involved in the defense system.Morus sp.) and analyzed their properties. LA-a and LA-b have molecular masses of approximately 50 and 46 kDa, respectively, and are abundant in the soluble fraction of latex. Western blotting analysis suggested that they share sequence similarity with each other. The sequences of LA-a and LA-b, as determined by Edman degradation, showed chitin-binding domains of plant chitinases at the N termini. These proteins showed small but significant chitinase and chitosanase activities. Lectin RCA120 indicated that, unlike common plant chitinases, LA-a and LA-b are glycosylated. LA-a and LA-b showed insecticidal activities when fed to larvae of the model insect Drosophila melanogaster.We purified latex abundant protein a and b (LA-a and LA-b) from mulberry (Our results suggest that the two LA proteins have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin. Hevea brasiliensis) and rubber fig (Ficus elastica), insoluble latex particles are a well-known commercial source of natural rubber [Arabidopsis, rice, wheat, and tobacco, do not have laticifers. However, they have been reported in about 12 500 plant species in 22 families, including monocots and dicots, and are estimated to exist in up to 20 000 species in 40 families [Laticifer cells of plants are unique in shape, development and function. These cells form long tubular or branched structures that run through the plant body, and large amounts of cytoplasm are exuded when the plant body is cut. The cytoplasm is a sticky white fluid called latex, and in the case of the para rubber tree (l rubber . LaticifPapaver bracteatum), narcotic alkaloid morphine in the opium poppy (Papaver somniferum), and insecticidal compounds such as the glycosidase inhibitors 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ) in mulberry [Carica papaya) and wild fig  is toxic to caterpillars of herbivorous insects [One important feature of laticifers is that they contain various toxic compounds in the latex; for example, the neurotransmitter dopamine in the Persian poppy  with that of hen egg lysozyme. In that study, the activity of 2 isoforms was 100 times higher than that of hen egg lysozyme, and the activity of 5 isoforms was 2.5-10 times higher. We also compared the activity of LA-a and LA-b with that of hen egg lysozyme. At pH 5, chitinase activity of LA-a and LA-b was 1/10 and 1/7, respectively, of that of hen egg lysozyme at pH 5 (data not shown). Compared to the orange chitinases, LA-a and LA-b thus had markedly lower chitinase activity.The sequence similarity between the chitin-binding domains of known chitinases and the N-terminal sequences of LA-a and LA-b suggests that they are also chitinases. We therefore examined the enzyme activities of LA-a and LA-b. When using carboxymethylated (CM) chitin labeled with remazol brilliant violet (RBV) as a substrate, LA-a and LA-b showed chitinase activity Figure . When acet al.  have comet al. [LA-a and LA-b also were able to hydrolyze chitosan, which is partly deacetylated and therefore solubilized chitin. Chitosanase activity was tested at a pH range of 3-7, and maximum activity was observed at pH 5 Figure . At pH 5et al. , chitosaThese results indicated that LA-a and LA-b had chitinase and chitosanase activities, although the activities were relatively low compared to those of orange chitinases.D. melanogaster. D. melanogaster has some advantages over other insects; it is easy to rear, and its small size and short life cycle reduces food requirements for the assay. In addition, because D. melanogaster does not eat mulberry, it was not expected to have any existing mechanism for overcoming the potential toxicity of mulberry protein. When 2-day-old larvae (second instar) were fed an artificial diet that contained 0.1% (w/w) LA-a or LA-b, very few developed into pupae over 6 days of incubation  and Eri silkworm (Samia richini), but not on mulberry specialist silkworm (Bombyx mori). Similarities between LA-a and LA-b proteins and MLX56, with respect to N-terminal amino acid sequence and electrophoretic mobility in SDS-PAGE, suggest that they are the same or similar proteins. However, contrary to our results, MLX56 has been reported to have no chitinase activity [3), a common substrate for plant chitinase assay, was used (data not shown). Substrate specificity of LA proteins and MLX56 could be different from that of common plant chitinases.In a recently published study  on mulbeactivity . Enzyme D. melanogaster and other insects. In addition to external body surfaces, the inside of the mouthpart is also a possible target of LA-a and LA-b because the pH inside the mouthpart is expected to be near that of crude extract of mulberry tissues  and to be optimal for the chitinase activity of LA proteins.Chitin, a substrate of chitinase, forms the cuticle of the exoskeleton in insects, and should be the target of LA-a and LA-b in the larvae of Populas sp.) chitinase inhibited development of Colorado potato beetle (Leptinotarsa decemlineata) larvae, and the amount of chitinase in the leaves was >0.3% (w/w) [Manduca sexta) included at 2% in an artificial diet showed 100% mortality of larvae of merchant grain beetles (Oryzaephilis mercator) [Heliothis virescens) larvae [Plant chitinase is well known to have antifungal activity, but only a few studies have investigated its toxicity to insects. It has been reported that transgenic tomato that accumulated poplar (3% (w/w) . Two papercator) , and tra) larvae .The insecticidal activity of LA proteins might not be caused simply by chitinase activity. It could be that a domain of LA proteins with unidentified biochemical activity cooperates with the chitinase domain to enhance insecticidal activity. Estimated molecular masses of LA-a and LA-b (50 and 46 kDa) were higher than those of common plant chitinases (20-40 kDa). This is at least partly caused by glycosylation, but it also suggests that the proteins might have another functional domain and biochemical function, working in cooperation with chitinase activity for efficient toxicity. Further analysis is needed to understand the molecular mechanism of insecticidal activity of LA-a and LA-b.D. melanogaster in this study is that this insect does not eat mulberry and thus is not expected to overcome potential toxicity of LA proteins. It would be interesting to know to which species of insect other than D. melanogaster  the LA proteins are toxic. Some insect species including silkworm would be tolerant to LA proteins, and it would be interesting to investigate the mechanism by which they can overcome the toxicity of the LA proteins.Unlike some other insects that eat leaves of latex-producing plants, silkworm eats mulberry leaves without removing the latex by cutting or trenching veins  were abundantly accumulated in latex of mulberry and suggested that they have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin.Morus alba L. cv. Minamisakari) was maintained at the Center for Bioresource Field Science, Kyoto Institute of Technology.Mulberry  and dialyzed against the same buffer overnight, using a dialysis membrane with a molecular weight cut-off of 25 kDa . The sample was diluted to 50 ml and loaded onto a CM-Cellulofine C-200  cation-exchange column (26 mm i.d. × 100 mm) equilibrated with buffer B. Protein fractions that contained LA-a and LA-b were eluted with buffer B that contained 0.2 M KCl at a flow rate of 1.0 ml min-1. Eluted protein solution was brought to 30% saturation with ammonium sulfate, and hydrophobic interaction chromatography was carried out on a HiLoad 16/10 phenyl sepharose HP column  equilibrated with buffer B that contained ammonium sulfate at 30% saturation. The column was developed with a linear gradient of 30-0% saturation of ammonium sulfate at a flow rate of 2 ml min-1. Fractions that contained LA-b were desalted twice by gel filtration chromatography using PD10  and 10DG  column, and further purified on a UNOS1 cation-exchange column (Bio-Rad) eluted with a linear gradient of 0-0.5 M NaCl in buffer B at a flow rate of 3.0 ml min-1. Purified LA-a and LA-b proteins were concentrated with Microcon YM-10  to 1.0 mg ml-1, frozen with liquid nitrogen, and stored at -80°C. All purification steps were performed at 4°C. All chromatography steps were carried out using a BioLogic Duo-Flow chromatography system (Bio-Rad). Protein concentrations were determined according to the equation: protein concentration (mg ml-1) = 1.45 A280 - 0.74 A260 [Latex exuded from the cut petiole of mulberry was mixed rapidly with an equal volume of buffer A  supplemented with 0.1% (v/v) β-mercaptoethanol, frozen in liquid nitrogen, and stored at -80°C. Six milliliters of the latex/buffer A mixture was diluted 4 times with buffer A and centrifuged at 18 000 .74 A260 .et al. [A rat polyclonal antibody was raised against purified LA-a by a standard procedure. For western blotting, protein samples were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane using the buffer system of Kyhse-Anderson . The memet al. . A mixtu-1. Eluted proteins were concentrated by evaporation and analyzed by automated protein sequencer PPSQ-21A .Cysteine residues of proteins were alkylated by treatment with iodoacetamide according to Coligan et al. . ProteinProtein samples were separated by SDS-PAGE and the gel was stained, first with Pro-Q Emerald 300 to detect glycosylated proteins, and then with SYPRO Ruby stain  for total proteins, according to the manufacturer's instructions. As a control, CandyCane glycoprotein molecular weight standards (Invitrogen) were used. For the lectin blot, protein samples were separated by SDS-PAGE and electroblotted onto a PVDF membrane as described above. The membrane was reacted with biotinylated RCA120 , and subsequently with alkaline phosphatase-conjugated streptavidin. A mixture of BCIP and NBT was used for color development. Fifteen other lectins  were purchased from Seikagaku Kogyo .et al. [2HPO4 buffers (pH 5-8). Hydrolyzed chitin was recovered and measured by absorbance at 550 nm. Activity was expressed as Δ550 nm g protein-1 min-1. Fluorometric chitosanase assay was performed using soluble chitosan  as a substrate according to Osswald et al. [2HPO4 buffers (pH 3-7). Controls consisted of the complete reaction mixtures stopped immediatedly after addition of enzyme, as described by Osswald et al. [Colorimetric chitinase assay was performed using CM-chitin-RBV  as a substrate, according to Mayer et al. , except d et al. , except d et al. . The reaDrosophila melanogaster (Canton-S strain) was reared at 25°C on standard yeast agar medium. To test insecticidal activity of LA-a and LA-b proteins, 15 2-day-old larvae (second instar) were placed in a vial that contained 200 mg Formula 4-24 instant Drosophila medium , rehydrated with 0.8 ml PBS supplemented with 1 mg LA protein and 10 mg Bacto Yeast Extract  as a nitrogen source. This accounted for 0.1% (w/w) LA protein. For control experiments, the diet was supplemented with the same amount of bovine serum albumin (BSA) or no protein instead of LA protein. The vials were incubated at 25°C.D. melanogaster experiment. FK, AK and YI carried out all purification and biochemical analysis. SK designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.KK helped with Edman degradation of LA proteins. ST helped with preparation of antibody. MY helped with the